Hwa-Jee Chung

Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites

Methyl jasmonate (MeJA) treatment increases the levels of plant secondary metabolites, including ginsenosides, which are considered to be the main active compounds in ginseng (Panax ginseng C.A. Meyer). To create a ginseng gene resource that contains the genes involved in the biosynthesis of secondary metabolites, including ginsenosides, we generated 3,134 expression sequence tags (ESTs) from MeJA-treated ginseng hairy roots. These ESTs assembled into 370 clusters and 1,680 singletons. Genes yielding highly abundant transcripts were those encoding proteins involved in fatty acid desaturation, the defense response, and the biosynthesis of secondary metabolites. Analysis of the latter group revealed a number of genes that may be involved in the biosynthesis of ginsenosides, namely, oxidosqualene cyclase (OSC), cytochrome P450, and glycosyltransferase. A novel OSC gene was also identified by this analysis. RNA gel blot analysis confirmed that transcription of this OSC gene, along with squalene synthase (SS) and squalene epoxidase (SE) gene transcription, is increased by MeJA treatment. This ginseng EST data set will also provide important information on the genes that are involved in the biosynthesis of other secondary metabolites and the genes that are responsive to MeJA treatment.

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.

Production of Human Serum Albumin in Chloroplast-Transformed Tobacco Plants

Human serum albumin (HSA) is the most abundant protein in plasma and is the most often used intravenous protein in many human therapies. However, HSA is currently extracted only from plasma because commercially feasible recombinant expression systems are not available. This study attempted to develop an efficient system for recombinant HSA production by chloroplast transformation of tobacco. A HSA cDNA was isolated from a cDNA library constructed with human liver tissue. Chloroplast transformation vectors were constructed by introducing various regulatory elements to HSA regulatory sequences. Vectors were delivered by particle bombardment into leaf explants and chloroplast-transformed plants were subsequently regenerated into whole plants. Southern blot analysis confirmed that the HSA cDNA was incorporated between rps12 and orf70B of the chloroplast genome as designed. Western blot analysis revealed that hyper-expression and increasing the stability of HSA were achieved by modification of the regulatory sequences using the psbA5`UTRs in combination with elements of the 14 N-terminal amino acids of the GFP and the FLAG tag. However, only plants transformed with the vector containing all of these elements were able to accumulate HSA.

Chloroplast Genetic Transformation in Higher Plants: An Encounter Between Prokaryote and Eukaryote

Chloroplasts are believed to be descended from certain cyanobacteria, which were taken up by phagocytosis into a host cell and lived there in a symbiotic relationship. In contrast to the current static concept on the chloroplast genome, its dynamism has been recently demonstrated: the chloroplast genome is active in intramolecular homolgous recombination, producing subgenomic circles when it obtains homolgous sequences via genetic transformation. Chloroplast tranformation in higher plants provides many advantages over nuclear transformation that include higher expression levels of transgenes, polycistronic expression of transgenes, and maternal transmission of transgenes. Tobacco has been used as a model for chloroplast genetic transformation. However, it is recently possible to transform the chloroplasts of other major food and economic crops including rice, soybean, and cotton. Chloroplast-transformed crops will be able to replace bioreactors using microorganisms for production of value-added proteins in future.

Current status on plant molecular farming via chloroplast transformation

Abstract Chloroplast transformation in higher plants offers many attractive advantages over nuclear transformation, including a high-level accumulation of foreign proteins, multi-gene expression in single transformation event via transgene stacking in operons and no position effect due to site-specific integration of transgenes by homologous re-combination. Most importantly, chloroplast transgenic plants are eco-friendly because their transgenes are maternally in-heritance in most crop plants. However, chloroplast trans-formation system has limited success in crops alike nuclear transformation. In the past two decades, great progress has been made to overcome the limitations of chloroplast transformation, thus expending chloroplast bioreactor to several important crops including soybean, carrot, lettuce, and oilseed. Therefore, it has become possible that chloro-plast transformation of crops can be used not only for the improvement of agronomic traits, but also for the production of vaccines and high valuable therapeutic proteins in pharmaceutical industry.

Mass production and application of activation tagged hairy root lines for functional genomic of secondary metabolism in ginseng

Abstract Activation tagging that uses T-DNA vectors con-taining multimerized transcriptional enhancers from the cau-liflower mosaic virus (CaMV) 35S gene is a powerful tool to determine gene function in plants. This approach has been successfully applied in screening various types of mutations and cloning the corresponding genes. We generated an acti-vation tagged hairy root pool of ginseng ( Panax ginseng C.A. Meyer) in an attempt to isolate genes involved in the bio-synthetic pathway of ginsenoside (triterpene saponin), which is known as the major active ingredient of the root. Quanti-tative and qualitative variation of ginsenoside in activation tagged hairy root lines were profiled using LC/MS. Metabolic profiling data enabled selection of a specific hairy root line which accumulated ginsenoside at a higher level than other lines. The relative expression level of several genes of tri-terpene biosynthetic pathway in the selected hairy root line was determined by real time RT-PCR. Overall results suggest that the activation tagged ginseng hairy root system described in this study would be useful in isolating genes involved in a complex metabolic pathway from genetically intractable plant species by metabolic profiling.

Mass Production of Gain-of-Function Mutants of Hair Roots in Ginseng

본 연구는 아그로박테리움 공동배양법을 이용한 기능획득 인삼 모상근의 대량생산을 위한 조건 확립에 대한 것이다. 일반적으로, 인삼과 같이 형질전환을 통한종자의 확보가 어려운 식물에서는 loss-of-function을 이용한 기능유전체 연구에 한계가 있다. 한편, 유전자의 기능을 활성화시키는 방법 (gain-of-function)인 activation tagging 기술은 이러한 문제점을 극복할 수 있는 대안이 될 수 있으며, Agrobacterium rhizogenes를 이용한 모상근 생산 시스템은 대량의 돌연변이체를 안정적으로 용이하게 얻을 수 있다는 점에서 최적의 시스템이라고 할 수 있다. 본 연구에서는 activation-tagging된 효율적인 형질전환 모상근 생산에 있어서의 최적의 아그로박테리움 균주 및 인삼조직, 배지조성 등에 대한 조건을 확립하였으며, 다양한 배지에서의 형질전환 모상근의 생장률 및 분지율, 표현형 등을 조사하였다. 엽병 절편을 activation-tagging vector pKH01을 가지고 있는 A. rhizogenes R1000와 공동배양하였을 때 배양 4주후 85.9%의 빈도로 모상근이 생산되었다. 모상근의 최대 생장과 분지도를 나타내는 배양조건을 조사한 바 엽병절편을 1/2 SH 배지에서 4주 배양하였을 때 왕성하게 생장하였으며 2.6의 분지도를 보여주었다. 최종적으로 1,989개체의 독립적인 형질전환 모상근 line을 생산하였으며, 이들 모상근 line은 인삼 진세노사이드 생합성 관련 유전자의 발굴 및 기능해석에 유용하게 쓰일 것이다. 【This study describes conditions for the mass production of activation-tagged mutant hairy root lines of ginseng by cocultivation with Agrobacterium rhizogenes. Because it is not currently possible to produce progeny from transgenic ginseng, a loss-of-function approach for functional genomics cannot be appliable to this species. A gain-of-function approach is alternatively the choice and hairy root production by cocultivation of A. rhizogenes would be most practical to obtain a large number of mutants. Various sources of explants were subjected to genetic transformation with various strains of A. rhizogenes harboring the activation-tagging vector pKH01 to determine optimum conditions for the highest frequency of hairy root formation on explants. Petiole explants cocultivated with A. rhizogenes R1000 produced hairy roots at a frequency of 85.9% after 4 weeks of culture. Conditions for maximum growth or branching rate of hairy roots were also investigated by using various culture media. Petiole explants cultured on half strength Schenk and Hildebrandt medium produced vigorously growing branched roots at a rate of 2.6 after 4 weeks of culture. A total of 1,989 lines of hairy root mutants were established in this study. These hairy root lines will be useful to determine functions of genes for biosynthesis of ginsenosides.】

Production of stable chloroplast-transformed plants in potato (Solanum tuberosum L.)

Chloroplast genetic engineering of higher plants offers several unique advantages compared with nuclear genome transformation, such as high levels of transgene expression, a lack of position effect due to site-specific transgene integration by homologous recombination, multigene engineering in a single transformation event and reducing risks of gene flow via pollen due to maternal inheritance. We established a reproducible chloroplast transformation system of potato using a tobacco specific plastid transformation vector, pCtVG (trnI-Prrn-aadA-mgfp-TpsbA-trnA). Stable transgene integration into chloroplast genomes and the homoplasmic state of the transgenome were confirmed by PCR and Southern blot analyses. Northern, immunoblot analysis, and GFP fluorescence imaging revealed high expression and accumulation of GFP in the plastids of potato leaves. This system would provide new opportunities for genetic improvement and mass production of value added foreign proteins in this crop.

Gene Silencing Induced by Cytosine Methylation in Transgenic Tomato

토마토 형질전환체에서 NPTII 및 TPSP 유전자의 발현양상을 조사하였다. 4개 line 토마토 형질전환체가 선발배지에서 kanamycin에 저항성을 보이며 생산되었다. 그러나, 1번 line과 11번 line의 후대에서 NPTII 유전자의 발현이 억제되었으며, Kanamycin 저항성을 보이지 못했다. Southern 분석 결과, 1번 11번 line은 여러 copy의 외래 유전자를 가지고 있었으며, 2번, 10번 line은 1-2 copy의 외래유전자를 가지고 있었다. 형질전환 line 11번은 methylation sensitive 제한효소처리 후 DNA methylation 분석 결과, TPSP coding부위 및 도입유전자의 발현을 조절하는 CaMV35S 프로모터 주위에서 CpG methylation이 축적되었음이 확인되었다. 따라서 여러 copy의 외래유전자를 가진 토마토 형질전환 line 11번에서 NPTII 유전자 및 TPSP유전자의 발현이 억제된 것은 transcriptional gene silencing 기작에 의한 것으로 추정되었다. 【Transgene expression was analyzed in tomato plants. Four lines of neomycin phosphotransferase II gene (NPTII) and the trehalose biosynthetic fusion gene (TPSP) transformed

Feasibility of Determining the Ripeness of Strawberry Fruit Flesh by Fourier Transform Infrared Spectroscopy

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